Graft versus host disease (GVHD) is a life-threatening complication of allogeneic hematopoietic stem cell transplantation (HSCT). GVHD prophylaxis is commonly based on either calcineurin inhibitors or post-transplant cyclophosphamide (PTCy). PTCy is increasingly employed for prevention of GVHD with improved overall outcomes. However, GVHD continues to be a significant problem and conventional chemotherapeutic toxicities of PTCy remain a concern. We have recently found that activated T cells display very strong DNA damage response (DDR) markers, and that small molecule-mediated inhibition of selected proteins in the DDR signaling cascade constitutes a novel, effective, and low toxicity method for deleting unwanted T cell responses. We are now seeking to investigate this therapy in allogeneic HSCT and preliminary studies indicated that combination DDR-based therapy, which we term `cell cycle checkpoint release' (CCCR) therapy, is highly effective at preventing GVHD in a murine model. Our work has led to our central hypotheses, that: 1) T cells initiating/ driving GVHD in mice and humans can be efficiently targeted with CCCR therapy with less off target toxicity than conventional DNA damaging agents such as cyclophosphamide, and 2) Because of its improved therapeutic index, episodic CCCR would be effective and tolerable therapy for established GVHD. We propose to test this hypothesis by first defining the efficacy of CCCR in multiple models as either prophylaxis or treatment of established GVHD. Next we will closely assess the toxicities of this approach, in comparison to PTCy. Finally, we will assess cells from patient undergoing HSCT to look for evidence of ongoing DDR and sensitivity to CCCR in activated T cells associated with clinical GVHD. Application of targeted DDR-based approaches is ideal for transplantation as the kinetics of allogeneic rejection/GVHD can be defined in clinical contexts. Successful application of targeted DDR-based approaches such as CCCR to transplantation would alter clinical paradigms and improve outcomes for patients undergoing transplantation.